Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb. The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. See generally, Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990.
Recombinant adenoviruses have advantages for use as transgene expression systems, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (see e.g., Berkner, K. L., Curr. Top. Micro. Immunol., 158:39-66 (1992); Jolly D., Cancer Gene Therapy, 1:51-64 (1994)).
Adenovirus vectors can accommodate a variety of transgenes of different sizes. For example, about an eight (8) kb insert can be accommodated by deleting regions of the adenovirus genome dispensable for growth (e.g., E3). Development of cell lines that supply non-dispensable adenovirus gene products in trans (e.g., E1, E2a, E4) has allowed insertion of a variety of transgenes throughout the adenovirus genome (see e.g. Graham, F. L., J. Gen. Virol., 36:59-72 (1977); Imler et al., Gene Therapy, 3:75-84 (1996)). For example, the p53, dystrophin, erythropoietin, ornithine transcarbamylase, adenosine deaminase, interleukin-2, .alpha.1antitrypsin, thrombopoietin, and cytosine deaminase genes have all been individually inserted into the adenovirus genome for making expression vectors.
The natural tropism of adenoviruses for respiratory tract cells has made them attractive gene therapy vectors for the treatment of cystic fibrosis (CF): the most common autosomal recessive disease in Caucasians. In CF, mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene disturbs cAMP-regulated C1.sup.- channel function, resulting in pulmonary dysfunction. The CFTR gene has been introduced into adenovirus vectors to treat CF in several animal models and human patients. Particularly, studies have shown that adenovirus vectors are fully capable of delivering CFTR to nasal epithelia of CF patients, as well as the airway epithelia of cotton rats and primates. See e.g., Zabner et al., Nature Genetics, 6:75-83 (1994); Rich et al., Human Gene Therapy, 4:461-476 (1993); Zabner et al., Cell, 75:207-216 (1993); Zabner et al., Nature Genetics, 6:75-83 (1994); Crystal et al., Nature Genetics, 8:42-51 (1994); Rich et al., Human Gene Therapy, 4:461-476 (1993).
Importantly, recent studies have demonstrated that it is possible to restore a functioning chloride ion channel in CF patients by providing an adenoviral vector encoding CFTR to airway epithelia cells (Zabner et al., J. Clin. Invest., 97:1504-1511 (1996)).
However, in vitro and in vivo studies have pointed to opportunities to further improve such vectors. For example, transgene expression from adenovirus vectors is often transient. Persistent transgene expression is highly desirable in gene therapy settings, especially those seeking to alleviate chronic or hereditary disease in mammals. At least some of the limitations are due to induction of a cell-mediated immune response against infected cells. In particular, cytotoxic T lymphocytes (CTLs) have been detected against antigenically expressed viral proteins encoded by adenovirus vectors, even though such vectors are replication defective. CTLs have also been detected against immunogenic transgene products. Cytotoxic T lymphocytes have the potential destroy or damage cells harboring the adenovirus vectors, thereby causing loss of transgene expression. Cell destruction can also cause inflamation which is also detrimental to the tissues involved. The cell-mediated immune response can pose a potentially serious obstacle to therapies requiring high dosages, which are likely to elicit more potent immune responses. See J. Kaplan et al., Human Gene Therapy 8:45-56 (1997); Y. Yang et al., Proc. Nat. Acad. Sci. 91:4405-11(1994); Y. Yang et al., J. Virol. 70:7202 (1996).
Various strategies have been used to minimize cell-mediated immune responses induced by adenovirus vectors. Generally, the strategies include modulation of the host immune response itself or engineering adenovirus vectors with a decreased capacity to induce immune responses.
For example, co-administration of immunosuppressive agents and adenovirus vectors have been reported to prolong persistence of transgene expression (Fang et al., Hum. Gene Ther., 6:1039-1044 (1995); Kay et al., Nature Genetics, 11:191-197 (1995); Zsellenger et al., Hum. Gene Ther., 6:457-467 (1995)).
In another approach, modification of adenovirus genome sequences in recombinant vectors has been used in attempts to decrease recognition of the vector by the immune system (see e.g., Yang et al., Nature Genetics, 7:362-369 (1994); Lieber et al., J. Virol., 70:8944-8960 (1996); Gorziglia et al., J. Virol., 70:4173-4178 (1996); Kochanek et al., Proc. Natl. Acad. Sci. USA, 93:5731-5736 (1996); Fisher et al., Virology, 217:11-22 (1996)).
The choice of promoter or transgene may also influence persistence of transgene expression from adenovirus vectors (see e.g., Guo et al., Gene Therapy, 3:801-802 (1996); Tripathy et al., Nature Med., 2:545-550 (1996)).
Persistence of transgene expression from adenovirus vectors has been reported to be influenced by the adenovirus E3 gp19K protein. That protein can complex with MHC Class I molecules in the endoplasmic reticulum, thereby preventing both cell surface presentation of viral antigens and killing of transduced cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol., pp. 437-443, (1994)). However, approaches based on that knowledge have only achieved limited success.
Another problem which has faced researchers who are attempting to utilize viral vectors for gene therapy has been the size of the heterologous DNA which can be inserted into the modified viral genome. Early work involving insertion of heterologous genes in the area of E1 deletion resulted in vectors which were difficult to produce in sufficient quantities to permit continued clinical testing. See, e.g., D. Armentano et al., Hum. Gene Ther., 6:1343 at 1344 (1995). While it is possible to possible to produce viral vectors which contain adenoviral DNA that is longer than the wild type genome length, the ability to replicate such vectors can decrease precipitously when the wild type genome length is substantially exceeded.
Accordingly, there is a need to develop and produce transgene expression vectors that have a genome whose size permits packaging that provides persistent transgene expression and minimizes cell-mediated immune reactions against cells containing the vectors. Such vectors would have a variety of uses, including use as gene transfer vectors in gene therapy.